CN101250572B - Method for extracting pig blood antibiotic peptide - Google Patents
Method for extracting pig blood antibiotic peptide Download PDFInfo
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- CN101250572B CN101250572B CN2007101764775A CN200710176477A CN101250572B CN 101250572 B CN101250572 B CN 101250572B CN 2007101764775 A CN2007101764775 A CN 2007101764775A CN 200710176477 A CN200710176477 A CN 200710176477A CN 101250572 B CN101250572 B CN 101250572B
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Abstract
The invention discloses a method for extracting porcine blood antibacterial peptide, which comprises following steps: firstly, dissolving porcine blood powder in buffer solution, then, adding papain into mixture to carry out enzymatic hydrolysis, adjusting the pH value of enzymolysis liquid to be 3.5-4.5, secondly, adding chloroform and sodium bisulfite into the enzymolysis liquid to decolorize, and then adjusting the pH value of the sodium bisulfite which is decolorized to be 6.0-7.0 and to form crude extracts of the porcine blood antibacterial peptide. Aiming at obtaining finished goods of the porcine blood antibacterial peptide, following purification steps are carried out to the crude extracts: firstly, freezing and drying the crude extracts of the porcine blood antibacterial peptide, enabling the crude extracts to pass through a gel-type cation exchange column, eluting the exchange column, collecting eluent, and secondly, freezing and drying the eluent in vacuum to form porcine blood antibacterial peptide. The method of the invention provides a new approach and a new method for producing antibacterial peptide. The method of the invention has the characteristics of simple operation, high separation extraction rate and low cost, and is suitable for separating and purifying the antibacterial peptide from porcine blood with comparatively large amount.
Description
Technical field
The present invention relates to a kind of method of extracting antibacterial peptide, particularly about a kind of method of extracting pig blood antibiotic peptide.
Background technology
At present, because antibiotic a large amount of abuses, the problem of traditional antibiotic resistance more and more causes people's attention, and the novel antibacterial medicine that seek a kind of noresidue, has no drug resistance has become the task of top priority.Antibacterial peptide is because its fungistatic effect and extensive and stable biological activity efficiently cause people's interest and concern gradually.Antibacterial peptide has wide spectrum, antimicrobial acivity efficiently, can also promote growth of animal, the acquired immunity of induced animal.And the antibiotic mechanism of its uniqueness can not make the microorganisms resistance, and noresidue in body has the good application development prospect, and the application of antibacterial peptide may bring the new revolution of an antimicrobial strategy.
China is pig industry big country, and pig blood is the waste of pig slaughtering processing, how to make the high-valued utilization of this generation of waste materials, has many-sided significances such as waste processing and utilization and public health.Relevant research confirms to have the polypeptide of strong anti-microbial activity in the pig blood, so development research is a kind ofly extracted from pig blood, the maturation method of purifying antibacterial peptide, can achieve many things at one stroke.And present technological method and the product that from pig blood, extracts antibacterial peptide that at home and abroad yet there are no.
Summary of the invention
The purpose of this invention is to provide a kind of method of extracting pig blood antibiotic peptide.
The present invention extracts the method for pig blood antibiotic peptide, may further comprise the steps:
1) the pig blood meal is dissolved in the damping fluid, add papoid then and carry out enzymolysis in mixed solution, the pH that adjusts enzymolysis solution is 3.5～4.5;
2) chloroform and sodium bisulfite are joined in the described enzymolysis solution decolour, adjust pH value to 6.0～7.0 of decolouring back enzymolysis solution then, become the crude extract of pig blood antibiotic peptide.
Described crude extract is carried out following purification step, can obtain the pig blood antibiotic peptide highly finished product of purifying:
1) crude extract of the described pig blood antibiotic peptide of lyophilize, and described crude extract crossed the gel type cation exchange column, exchange column is carried out wash-out, collect elutriant;
2) described elutriant is carried out vacuum lyophilization, become pig blood antibiotic peptide.
In said process, damping fluid can be that pH=7.0, concentration are Sodium phosphate dibasic-citric acid mixed solution of 0.05mmol/L.The pig blood meal is 100: 1～3 with the ratio of the parts by weight of described papoid.The temperature of enzymolysis is 65-75 ℃, and the time of enzymolysis is 8～10h.
In the decolorization, described chloroform is 2～3: 5 with the ratio of described enzymolysis solution volume parts; The whole mass percentage concentration of sodium bisulfite is 0.5%～1.0%.
The temperature of lyophilize pig blood antibiotic peptide crude extract is generally-50～-60 ℃, and freezing time is 24h～48h.
The gel type cation exchange column can be selected Sephadex G100 gel column; Elutriant can be the ammonium acetate of 0.2mol/L, and the speed of wash-out is 10.0mL/cm
2.h; The temperature of elutriant being carried out vacuum lyophilization is generally-50～-60 ℃, and the cryodesiccated time is 24h～48h.
Raw material pig blood meal of the present invention can be bought common pig blood meal finished product on market, also can be prepared in order to following method voluntarily, and the method for preparation may further comprise the steps:
1) fresh pig blood being carried out anti-freezing handles;
2) described pig blood is filtered, remove fibrous matter wherein;
3) carry out multigelation to filtering down thing again, the pig blood meal is made in lyophilize.
The present invention has studied the separating and purifying technology of the low molecular peptide class material in the pig blood, and the biologic activity of the peptide matters of separation and purification studied, the result shows: multiple peptide matters has the obvious suppression effect to common pathogenic bacterium such as Staphylococcus albus, streptococcus aureus, Pseudomonas aeruginosa, Salmonellas, intestinal bacteria, Aeromonas hydrophilas in the pig blood; The pig blood polypeptide also all has significant deactivation to avian infectious bronchitis virus (IBV), newcastle disease virus (NDV) and chicken infectivity bursa of Fabricius virus (IBDV).More meaningfully, the polypeptide that the present invention is separated to can improve body non-specific immune function, specific chicken immune serum antibody horizontal and Intestinal Mucosal Immunity function, and can improve the chick day weight gain, reduce the material anharmonic ratio, promote growing of chicken embryo and chick.Result of study shows: the pig blood antibiotic peptide that the inventive method is produced is a kind of novel green anti-microbial agents and immunomodulator, can be widely used in herding manufacturing enterprise, fodder production or fodder additives manufacturing enterprises such as pig slaughtering factory.
The present invention has the following advantages: 1, the inventive method has realized extracting the peptide material with strong anti-microbial activity from pig blood, provides a new approaches and methods for producing antibacterial peptide.2, the antibacterial peptide that adopts the inventive method to produce belongs to natural extract, and have natural, nontoxic, pollution-free, noresidue, have no side effect, characteristics such as high-efficiency antimicrobial, be a kind of novel green antimicrobial agents.3, the antibacterial peptide that adopts the inventive method to produce, have broad-spectrum antimicrobial, antiviral, do not produce resistance, function such as immunoregulation effect arranged.4, the inventive method is easy to operate, and required equipment is simple, separation and Extraction rate height.5, enrich in the raw material pig blood source of implementing the inventive method, cheap, can satisfy the needs of extensive in-depth processing, has very strong Application and Development promotional value.Raw material pig blood belongs to the waste of pig slaughtering processing, and the inventive method can make waste obtain high-valued utilization, so the present invention has important public hygienics meaning.
Description of drawings
Fig. 1 is the chromatography graphic representation of pig blood antibiotic peptide crude extract through Sephadex G100 gel column
Fig. 2 is the antibacterial experiment design sketch of pig blood antibiotic peptide elutriant to the chicken pasteurella multocida
Embodiment
1, collects 1L fresh pig blood from the slaughterhouse, then, consumption by 6g/L blood adds the trisodium citrate anti-freezing rapidly, use filtered through gauze then, remove fibrous matter wherein, carry out multigelation and get hemolysate (freeze thawing is 1-3 time between-20 ℃ and 4 ℃) filtering down thing, the pig blood meal is made in lyophilize.The purpose of this step is broken pig hemocyte, discharges oxyphorase.
The pig blood meal also can adopt commercially available finished product pig blood meal.
2, the pH that 2g pig blood meal is dissolved in 200ml is 7.0, concentration is Sodium phosphate dibasic-citrate buffer solution (Lv Shiming, the Tan Aijuan of 0.05mmol/L.Papoid is to the hydrolytic action of pig blood powder protein.The Guizhou agricultural sciences, 2001,29 (4): 6～7), in mixed solution, add the papoid (blood meal: enzyme=100: 1～get final product at 100: 3), under 70 ℃ of (65-75 ℃ all can) conditions, carry out enzymolysis 8～10h of 20mg then.PH with 6mol/L HCl adjustment enzymolysis solution is 3.5～4.5 then, to eliminate residual organized enzyme.
3, adding the chloroform of 2/5～3/5 enzymolysis solution volume and final concentration in enzymolysis solution is 0.5%～1.0% sodium bisulfite, and water-bath 30min under 40～60 ℃ of conditions decolours, separatory, gets supernatant liquor.This decolorization is repeated 3 times, take off for colourless to supernatant liquor.
4, drip 1mol/L NaOH, the enzymolysis solution after the decolouring is adjusted its pH value to 6.0～7.0, become the crude extract of pig blood antibiotic peptide.
5, adopt vacuum freeze drier, with the crude extract of pig blood antibiotic peptide carry out conventional vacuum freezing, (freezing temp is-50～-60 ℃ to drying, and be 24～48h) time of drying, makes pig blood antibiotic peptide crude extract dry powder.Get 5g crude extract dry powder then and place Sephadex G-100 gel ion exchange column, with the ammonium acetate elutriant of 0.2mol/L crude extract is carried out wash-out and separate, flow velocity is 10.0mL/cm
2.h, collect about 40mL elutriant.
As shown in Figure 1,1 elution peak appears in pig blood extract behind Sephadex G100 gel filtration chromatography.The component of each collection tube is got 20 μ L carry out bacteriostatic test.
When 6, collecting elutriant,, adopt the agarose diffusion method that the elutriant of collecting is detected, detect and collect the anti-microbial effect of liquid the chicken pasteurella multocida through the nucleic acid-protein detector.The result shows promptly have inhibition zone to occur from the 13rd pipe, and to the 23rd pipe inhibition zone disappearance, maximum inhibition zone appears at the 17-21 pipe.(Figure 2 shows that the situation of 1-18 pipe).
Merge elutriant, carry out vacuum lyophilization, become pig blood antibiotic peptide at-20 ℃ with bacteriostatic action.Under-20 ℃ of conditions, pig blood antibiotic peptide is preserved.
Embodiment 2, antibiotic, the antiviral test of pig blood antibiotic peptide
1, the antibacterial activity test of pig blood antibiotic peptide
Adopt agarose disperse method, the pig blood antibiotic peptide that embodiment 1 is extracted from pig blood carries out the anti-microbial activity detection.Have for the examination bacterium: chicken pasteurella multocida, the strain of chicken pathogenic Salmonellas natural separation, intestinal bacteria O
88, streptococcus aureus ACTT25923 strain, Pseudomonas aeruginosa ACTT27853 strain.
The measurement result of pig blood antibiotic peptide anti-microbial activity shows that pig blood antibiotic peptide is to chicken pasteurella multocida, chicken pathogenic Salmonellas, intestinal bacteria O
88, streptococcus aureus ACTT25923 strain, Pseudomonas aeruginosa ACTT27853 strain all have the obvious suppression effect, concrete outcome is as shown in table 1.
Table 1 pig blood antibiotic peptide is to the fungistatic effect of different strains
Remarks: " ++ ++ " more than the 13mm, " +++" 11--13mm, " ++ " 8--10mm, "+" 5--7mm,
Below "-" 5mm.
The result shows that pig blood antibiotic peptide all has stronger bacteriostatic action to Gram-negative bacteria and gram-positive microorganism.
2, the antiviral activity of pig blood antibiotic peptide experiment
Get 12 pieces of instar chicken embryos on the 10th, be divided into 3 groups at random.I group (NDV group) inoculation newcastle disease virus; The NDV that II group (ABP treatment group) inoculation is handled through pig blood antibiotic peptide of the present invention; III group (control group) inoculation sterile saline.Every piece of chick embryo allantoic cavity inoculation sample 0.1mL.After the allantoic cavity inoculation, every 12h according to egg once observes the chick embryo development situation.To 18 ages in days, cut open chicken embryo extremely, observe the chicken embryo and change, weigh, and get the freezing preservation of its allantoic fluid, carry out the mensuration of NDV hemagglutinative titer.
Experimental result is as follows:
(1) the cardinal principle pathology of chicken embryo
Behind the egg inoculation virus liquid the 4th day, the hypomotility of visible NDV group chicken embryo.Postvaccinal the 8th day, cut open inspection chicken embryo and observe, visible NDV group chick embryo yolk sac shrinks, and cyst membrane is easily torn, and allantoic fluid increases; Short and small embryo occurs, amnion thickens and sticks on the idiosome.And the chick embryo development of ABP treatment group and control group is normal.
(2) comparison of each treatment group chicken embryo weight
Wipe away dried chicken embryo with thieving paper, weigh, ABP treatment group chicken embryo weight and control group difference are not remarkable, and virus group chicken embryo weight is starkly lower than preceding two groups (as shown in table 2).
The comparison of chicken embryo weight between table 2 different treatment (
N=4)
| Grouping | The ABP treatment group | Control group | The NDV group |
| Chicken embryo weight/g | ?21.63±0.62a | 21.7±0.51a | 20.89±1.05b |
NDV hemagglutinative titer measurement result is as shown in table 3.
The hemagglutinative titer of NDV in table 3 chick embryo allantoic liquid (
N=4)
| Grouping | The ABP treatment group | Control group | The NDV group |
| Hemagglutinative titer (log2) | 0.75±0.5a | 0a | 3.25±0.5b |
Table 3 remarks: mean carries out the q check between group, indicates different alphabetical persons and represents difference extremely significantly (p＜0.01), indicates same letter person and represents significant difference (p＜0.05).
This shows that through the viral liquid that pig blood antibiotic peptide is handled, owing to the mutual neutralizing effect of viral and pig blood antibiotic peptide, the virulence of feasible virus is lost or weakened, and can not breed in a large number, thereby it is lower to show as viral hemagglutinative titer in the chicken embryo.Thereby illustrate that pig blood antibiotic peptide has stronger deactivation to virus.
Claims (8)
1. method of extracting pig blood antibiotic peptide may further comprise the steps:
1) the pig blood meal is dissolved in the damping fluid, add papoid then and carry out enzymolysis in mixed solution, the pH that adjusts enzymolysis solution is 3.5～4.5;
2) chloroform and sodium bisulfite are joined in the described enzymolysis solution decolour, adjust pH value to 6.0～7.0 of decolouring back enzymolysis solution then, become the crude extract of pig blood antibiotic peptide;
Described crude extract is carried out following purification step:
1) crude extract of the described pig blood antibiotic peptide of lyophilize, and described crude extract crossed the gel type cation exchange column carries out wash-out to exchange column with the ammonium acetate of 0.2mol/L, collects elutriant;
Described gel type cation exchange column is a Sephadex G100 gel column;
2) described elutriant is carried out vacuum lyophilization, become pig blood antibiotic peptide.
2. the method for claim 1, it is characterized in that: described damping fluid is that pH=7.0, concentration are Sodium phosphate dibasic-citric acid mixed solution of 0.05mmol/L.
3. the method for claim 1, it is characterized in that: described pig blood meal is 100: 1～3 with the ratio of the parts by weight of described papoid.
4. method as claimed in claim 3 is characterized in that: the temperature of described enzymolysis is 65-75 ℃, and the time of enzymolysis is 8～10h.
5. the method for claim 1, it is characterized in that: described chloroform is 2～3: 5 with the ratio of described enzymolysis solution volume parts; The whole mass percentage concentration of sodium bisulfite is 0.5%～1.0%.
6. the method for claim 1, it is characterized in that: the temperature of described lyophilize pig blood antibiotic peptide crude extract is-50～-60 ℃, freezing time is 24h～48h.
7. the method for claim 1, it is characterized in that: the speed of described wash-out is 10.0mL/cm
2.h; The temperature of described elutriant being carried out vacuum lyophilization is-50～-60 ℃, and the cryodesiccated time is 24h～48h.
8. the method for claim 1, it is characterized in that: the preparation of described pig blood meal may further comprise the steps:
1) fresh pig blood being carried out anti-freezing handles;
2) described pig blood is filtered, remove fibrous matter wherein;
3) carry out multigelation to filtering down thing again, the pig blood meal is made in lyophilize.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101375702B (en) * | 2008-09-12 | 2011-05-18 | 中国农业大学 | Hemoglobin polypeptide powder and preparation method thereof |
| CN103539850A (en) * | 2012-07-16 | 2014-01-29 | 拜宁生物科技股份有限公司 | Peptide separately separated from pig and use thereof |
| CN103431155A (en) * | 2013-09-16 | 2013-12-11 | 陆杰坤 | Production technology for enzymolysis and decoloration of spray-dried animal blood cells |
| CN104164467B (en) * | 2014-08-08 | 2017-01-11 | 中国农业大学 | Method for extracting antibacterial peptide from chicken blood |
| CN107929711B (en) * | 2017-12-01 | 2021-08-31 | 福建蓝昊肽生物科技发展有限公司 | Application of blood-derived enzymolysis peptide in preparation of medicine for inhibiting animal intestinal inflammation |
| CN108553635B (en) * | 2018-04-27 | 2021-10-12 | 武汉华腾济康生物科技有限公司 | Pig-derived antibacterial peptide PMAP-36 sustained-release capsule |
| CN108546728A (en) * | 2018-04-29 | 2018-09-18 | 中南民族大学 | A kind of method that porcine haemoglobin enzymolysis decolourizes and its application in preparation relieves fatigue drug and antitumor drug |
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| CN1858228A (en) * | 2006-03-28 | 2006-11-08 | 华南理工大学 | Process for preparing low molecular peptide and amino acid by biological enzymolysis of pig blood haematoglobin |
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| CN1858228A (en) * | 2006-03-28 | 2006-11-08 | 华南理工大学 | Process for preparing low molecular peptide and amino acid by biological enzymolysis of pig blood haematoglobin |
Non-Patent Citations (3)
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| 吴琼英 等.抗菌乳的制备及其初步纯化.《中国乳品工业》.2007,第35卷(第1期),15-18. |
| 吴琼英等.抗菌乳的制备及其初步纯化.《中国乳品工业》.2007,第35卷(第1期),15-18. * |
| 杨万根.酶解猪血红蛋白制备小肽方法的研究.《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》.2004,(第04期),B024-105. * |
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